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The yeast nuclear pore complex: composition, architecture, and transport mechanism.

Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, Chait BT - J. Cell Biol. (2000)

Bottom Line: Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins).This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC.Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York, NY 10021, USA. rout@rockvax.rockefeller.edu

ABSTRACT
An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.

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Localization of the tagged nucleoporins by immunoelectron microscopy. To estimate the position of the tagged nups within the NPC, we immunolabeled NEs from each protein A–tagged strain using a gold-conjugated antibody to visualize the tag. We selected labeled NPC images that had no obvious signs of damage or occlusion, and whose nucleocytoplasmic orientation could be unambiguously determined (Nehrbass et al. 1996). We chose only those NPCs sectioned perpendicular to the NE plane with a clearly visible double membrane. We selected a radius of 100 nm around the estimated center of each NPC as an excision limit and then created an aligned superimposed montage using 20 of the resulting excised NPC images (Strambio-de-Castillia et al. 1999). Scale bars are graduated in 10-nm intervals, with the horizontal bar centered on the cylindrical axis of the NPC and the vertical bar centered on the plane of NPC pseudo-mirror symmetry. The major features in each montage are diagrammed schematically on the lower right (C, cytoplasm; N, nucleoplasm). Montages from extraction conditions with the highest degree of specific labeling (averages ranging between 3–10 gold particles/NPC) are shown. They are arranged into those showing approximately symmetric labeling on both sides of the NPC, those with labeling biased towards one face, and those localized exclusively either to the cytoplasmic face or the nucleoplasmic face. NUP145N and NUP145C, NH2-terminal and COOH-terminal tagged fragments respectively of Nup145p (in all other figures and tables NUP145 refers only to the COOH-terminal tagged fragment).
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Figure 7: Localization of the tagged nucleoporins by immunoelectron microscopy. To estimate the position of the tagged nups within the NPC, we immunolabeled NEs from each protein A–tagged strain using a gold-conjugated antibody to visualize the tag. We selected labeled NPC images that had no obvious signs of damage or occlusion, and whose nucleocytoplasmic orientation could be unambiguously determined (Nehrbass et al. 1996). We chose only those NPCs sectioned perpendicular to the NE plane with a clearly visible double membrane. We selected a radius of 100 nm around the estimated center of each NPC as an excision limit and then created an aligned superimposed montage using 20 of the resulting excised NPC images (Strambio-de-Castillia et al. 1999). Scale bars are graduated in 10-nm intervals, with the horizontal bar centered on the cylindrical axis of the NPC and the vertical bar centered on the plane of NPC pseudo-mirror symmetry. The major features in each montage are diagrammed schematically on the lower right (C, cytoplasm; N, nucleoplasm). Montages from extraction conditions with the highest degree of specific labeling (averages ranging between 3–10 gold particles/NPC) are shown. They are arranged into those showing approximately symmetric labeling on both sides of the NPC, those with labeling biased towards one face, and those localized exclusively either to the cytoplasmic face or the nucleoplasmic face. NUP145N and NUP145C, NH2-terminal and COOH-terminal tagged fragments respectively of Nup145p (in all other figures and tables NUP145 refers only to the COOH-terminal tagged fragment).

Mentions: To estimate the position of each nup within the NPC from the immunoelectron microscopy labeling distributions, we first measured the distances of each gold particle (2,496 particles for the montages presented in Fig. 7) from both the cylindrical axis of the NPC and from its mirror plane (Kraemer et al. 1995). The resulting distributions of particle positions represent raw data uncorrected for cylindrical averaging, steric hindrance from the dense NPC core, and blurring effects arising from the distribution of antibody orientations. We developed a modeling procedure that corrected for each of these effects. Using this model, we calculated the expected cylindrically averaged and projected distributions for distance intervals of 2.5 nm, in both the R (position from the cylindrical axis) and Z (position from the mirror plane) directions, and determined the values of R and Z that gave the best fit to the raw experimental data for each nup. Rare examples of sagittally sectioned labeled NPCs allowed us to compare the accuracy of our R estimates with a directly measured R average figure; the two values were within 4 nm of each other, well within our estimated experimental error of ∼7 nm (data not shown). For symmetrically localized nups we pooled the distributions on both sides to increase the accuracy of our estimates.


The yeast nuclear pore complex: composition, architecture, and transport mechanism.

Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, Chait BT - J. Cell Biol. (2000)

Localization of the tagged nucleoporins by immunoelectron microscopy. To estimate the position of the tagged nups within the NPC, we immunolabeled NEs from each protein A–tagged strain using a gold-conjugated antibody to visualize the tag. We selected labeled NPC images that had no obvious signs of damage or occlusion, and whose nucleocytoplasmic orientation could be unambiguously determined (Nehrbass et al. 1996). We chose only those NPCs sectioned perpendicular to the NE plane with a clearly visible double membrane. We selected a radius of 100 nm around the estimated center of each NPC as an excision limit and then created an aligned superimposed montage using 20 of the resulting excised NPC images (Strambio-de-Castillia et al. 1999). Scale bars are graduated in 10-nm intervals, with the horizontal bar centered on the cylindrical axis of the NPC and the vertical bar centered on the plane of NPC pseudo-mirror symmetry. The major features in each montage are diagrammed schematically on the lower right (C, cytoplasm; N, nucleoplasm). Montages from extraction conditions with the highest degree of specific labeling (averages ranging between 3–10 gold particles/NPC) are shown. They are arranged into those showing approximately symmetric labeling on both sides of the NPC, those with labeling biased towards one face, and those localized exclusively either to the cytoplasmic face or the nucleoplasmic face. NUP145N and NUP145C, NH2-terminal and COOH-terminal tagged fragments respectively of Nup145p (in all other figures and tables NUP145 refers only to the COOH-terminal tagged fragment).
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Related In: Results  -  Collection

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Figure 7: Localization of the tagged nucleoporins by immunoelectron microscopy. To estimate the position of the tagged nups within the NPC, we immunolabeled NEs from each protein A–tagged strain using a gold-conjugated antibody to visualize the tag. We selected labeled NPC images that had no obvious signs of damage or occlusion, and whose nucleocytoplasmic orientation could be unambiguously determined (Nehrbass et al. 1996). We chose only those NPCs sectioned perpendicular to the NE plane with a clearly visible double membrane. We selected a radius of 100 nm around the estimated center of each NPC as an excision limit and then created an aligned superimposed montage using 20 of the resulting excised NPC images (Strambio-de-Castillia et al. 1999). Scale bars are graduated in 10-nm intervals, with the horizontal bar centered on the cylindrical axis of the NPC and the vertical bar centered on the plane of NPC pseudo-mirror symmetry. The major features in each montage are diagrammed schematically on the lower right (C, cytoplasm; N, nucleoplasm). Montages from extraction conditions with the highest degree of specific labeling (averages ranging between 3–10 gold particles/NPC) are shown. They are arranged into those showing approximately symmetric labeling on both sides of the NPC, those with labeling biased towards one face, and those localized exclusively either to the cytoplasmic face or the nucleoplasmic face. NUP145N and NUP145C, NH2-terminal and COOH-terminal tagged fragments respectively of Nup145p (in all other figures and tables NUP145 refers only to the COOH-terminal tagged fragment).
Mentions: To estimate the position of each nup within the NPC from the immunoelectron microscopy labeling distributions, we first measured the distances of each gold particle (2,496 particles for the montages presented in Fig. 7) from both the cylindrical axis of the NPC and from its mirror plane (Kraemer et al. 1995). The resulting distributions of particle positions represent raw data uncorrected for cylindrical averaging, steric hindrance from the dense NPC core, and blurring effects arising from the distribution of antibody orientations. We developed a modeling procedure that corrected for each of these effects. Using this model, we calculated the expected cylindrically averaged and projected distributions for distance intervals of 2.5 nm, in both the R (position from the cylindrical axis) and Z (position from the mirror plane) directions, and determined the values of R and Z that gave the best fit to the raw experimental data for each nup. Rare examples of sagittally sectioned labeled NPCs allowed us to compare the accuracy of our R estimates with a directly measured R average figure; the two values were within 4 nm of each other, well within our estimated experimental error of ∼7 nm (data not shown). For symmetrically localized nups we pooled the distributions on both sides to increase the accuracy of our estimates.

Bottom Line: Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins).This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC.Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York, NY 10021, USA. rout@rockvax.rockefeller.edu

ABSTRACT
An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.

Show MeSH
Related in: MedlinePlus